1. Field of the Invention
The present invention relates to methods for producing polypeptides. The present invention also relates to nucleic acid constructs, vectors, and host cells comprising consensus translational initiator sequences operably linked to nucleic acid sequences encoding polypeptides, and isolated consensus translational initiator sequences.
2. Description of the Related Art
The recombinant production of a heterologous protein in a fungal host cell, particularly a filamentous fungal cell such as Aspergillus, may provide for a more desirable vehicle for producing the protein in commercially relevant quantities.
Recombinant production of a heterologous protein is accomplished by constructing an expression cassette in which the DNA coding for the protein is placed under the expression control of a promoter, excised from a regulated gene, suitable for the host cell. The expression cassette is introduced into the host cell, usually by plasmid-mediated transformation. Production of the heterologous protein is then achieved by culturing the transformed host cell under inducing conditions necessary for the proper functioning of the promoter contained on the expression cassette.
Improvement of the recombinant production of proteins generally requires the availability of new regulatory sequences which are suitable for controlling the expression of the proteins in a host cell.
Kozak, 1981, Nucleic Acids Research 9: 5233-5252, proposed the following xe2x80x9cconsensusxe2x80x9d sequence for initiation of translation in higher eukaryotes:
Aa Acc AUG G
In this sequence, referred to as a xe2x80x9cconsensus Kozak,xe2x80x9d the most highly conserved nucleotides are the purines, adenine(A) and guanine (G), shown in capital letters above. Mutational analysis confirmed that these two positions have the strongest influence on initiation (Kozak, 1987, Molecular Cell Biology 7: 3438-3445). Kozak also determined that alterations in the sequence upstream of the consensus Kozak can effect translation (Kozak, 1986, Proceedings of the National Academy of Sciences USA 83: 2850-2854).
It is an object of the present invention to provide improved methods for producing a polypeptide in a fungal host cell using consensus translational initiator sequences.
The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a consensus translational initiator sequence foreign to the first nucleic acid sequence wherein the 3xe2x80x2 end of the consensus translational initiator sequence is immediately upstream of the initiator codon of the first nucleic acid sequence, and the consensus translational initiator sequence comprises the sequence 5xe2x80x2-NYCNNHCACC-3xe2x80x2 (SEQ ID NO. 1) wherein N is a nucleotide selected from the group consisting of adenine (A), guanine (G), cytosine (C), and thymine (T); Y is a cytosine (C) or thymine (T); and H is a nucleotide selected from the group consisting of adenine (A), cytosine (C), and thymine (T); and (b) isolating the polypeptide from the cultivation medium.
The present invention also relates to isolated consensus translational initiator sequences and to constructs, vectors, and fungal host cells comprising one or more of the consensus translational initiator sequences operably linked to a nucleic acid sequence encoding a polypeptide.